Probiotic strains for use in improving the enteric nervous system

ABSTRACT

The invention relates to the use of lactic acid bacteria, for use in modifying the enteric nervous system and more particularly in treating and/or preventing intestinal disorders such as constipation and/or irritable bowel disease.

FIELD

The present invention relates to compositions comprising strains oflactic acid bacteria for use in modifying the enteric nervous system.Such compositions are especially suitable to treat and/or preventintestinal disorders such as constipation and/or irritable boweldisease.

BACKGROUND

Irritable bowel syndrome (IBS) or spastic colon is a functional boweldisorder characterized by chronic abdominal pain, discomfort, bloating,and alteration of bowel habits in the absence of any detectable organiccause. In some cases, a low-grade gut inflammation was reported.Diarrhoea or constipation may predominate, or they may alternate(classified as IBS-D, IBS-C respectively). IBS may begin after aninfection (post-infectious, IBS-PI), a stressful life event or onset ofmaturity without any other medical indicators. In IBS, routine clinicaltests yield no abnormalities, though the bowels may be more sensitive tocertain stimuli, such as balloon insufflation testing.

IBS is a very common condition affecting approximately 15% of thepopulation at any one time. There are about twice as many women as menwith this condition. IBS is a source of chronic pain, fatigue and othersymptoms, and it increases a patient's medical costs, and contributes towork absenteism. Researchers have reported that the high prevalence ofIBS, in conjunction with increased costs produces a disease with a highsocietal cost. It is also regarded as a chronic illness and candramatically affect the quality of a sufferer's life.

A leading theory about the cause of IBS relates to the enteric nervoussystem (ENS). The enteric nervous system (ENS) is a subdivision of theperipheral nervous system (PNS) that directly controls gastro-intestinal(GI) functions and is embedded in the lining of the gastrointestinalsystem. It includes efferent neurons, afferent neurons, andinterneurons. The structural and physiological functioning of the ENS isperformed by glial cells (astrocytes). The ENS is organized into twomajor plexus with functional specific roles.

The myenteric plexus, located between the longitudinal and circularmuscle, contains neurones mainly involved in the control of intestinalmotility. Through intestinal muscles, the efferent or motor neuronscontrol peristalsis and churning of intestinal contents.

The motor neurons controlling motility are composed of two majorclasses:

-   -   excitatory myenteric neurons liberating acetylcholine (referred        to as Choline AcetylTransferase ImmunoReactive neurons        (“ChAT-IR” or “ChAT” or “ChAT neurones” or “ChAT nerves”) and/or        substance P (SP) for contractions, and    -   inhibitory myenteric neurons liberating nitric oxide (identified        as Nitric Oxide Synthase neurons (NOS-IR)) and/or Vaso-active        Intestinal Peptide (VIP) for relaxation.

Choline acetyltransferase EC 2.3.1.6 is an enzyme that is synthesizedwithin the body of a neuron and transferred to the nerve terminal. Therole of ChAT is to join Acetyl-CoA to choline, resulting in theformation of the neurotransmitter acetylcholine. Experimentally, effecton acetylcholine production is extrapolated from the determination ofthe number of ChAT neurons, typically an increase in the number of ChATnerves is indicative of an increase of acetylcholine.

The submucosal plexus, located between the circular muscle and themucosa, contains neurons mainly involved in the control of intestinalepithelial barrier (IEB) functions, such as paracellular permeability.In particular, activation of enteric neurones in the submucosal plexusdecreases paracellular permeability, via the liberation of VIP, whereasacetylcholine (Ach) increases paracellular permeability, setting thebasis of a fine ‘tuning’ of the IEBR permeability by the ENS. Thus,concerning the neuronal control of paracellular permeability, theincrease of VIP liberation by submucosal neurons increases IEB integritywhile the increase of submucosal plexus ChAT neurons decreases IEBresistance.

Although there is at current no cure for IBS, there are treatments whichattempt to relieve symptoms, including dietary adjustments, medicationand psychological interventions.

Probiotics, in particular strains of lactic acid bacteria, are reportedto be beneficial in the treatment and/or prevention of IBS. Examples ofsuch disclosures are WO 2007/036230, WO 03/010297, and WO 2009/080800.However, the bacterial strains are selected for their effect on theimmune system, on intestinal permeability or on the intestinalmicrobiota and not for their effect on improving the function of theENS. WO 2008/064489 discloses the use of probiotics to block anintermediate conductance calcium dependent potassium current resultingin an anti-inflammatory effect. WO 2007/132359 discloses the use ofLactobacillus and a cannabinoid receptor agonist and/or an opioidreceptor antagonist in relation to pain perception. WO 2006/032542discloses the use of Lactobacillus for analgesic purposes. Kamm et al,2004, Neurogastrointest. Motil 16: 53-60 disclosed effects of S.boulardii on decreasing calbindin-28 k (CALB) but not on other neuronalmarkers of the pig jejunum. Metugriachuk et al, 2006, Rejuvenation Res.9: 342-345 disclose that a symbiotic preparation on motility of smalland large intestine in old Wistar rats significantly increased themnyoelectric activity of small intestine and colon, an increased mRNAexpression of VIP, but no significant effect on VIP concentration.

Further research is therefore needed on individual strains of probioticbacteria with a beneficial effect on the ENS for use in IBS,constipation and/or other disorders.

SUMMARY OF THE INVENTION

The inventors employed a new model system for screening and selectingstrains of lactic acid bacteria and bifidobacteria which have animproved effect on the enteric nervous system (ENS). This model containsa (mono)layer of intestinal epithelial cells from human colon carcinomaand, on the basolateral side of the monolayer, a mixture of a primaryculture of enteric nervous system cells including neurones frommyenteric and submucosal plexus. Using this model the effects of foodgrade components, in particular strains of lactic acid bacteria andbifidobacteria, on the ENS could be assessed by measuring the effects ofapical or luminal addition of these components on the expression ofvaso-intestinal peptide (VIP) and/or ChAT releasing nerves on thebasolateral side.

This model therefore allowed to screen and to select new strains oflactic acid bacteria and bifidobacteria for use in improving thefunction of the enteric nervous system. Such strains improve intestinalmotility and peristalsis. With some strains the intestinal transit timecan be reduced, which can address some conditions such as constipation.With some strains the intestinal transit time can be increased, whichcan address some conditions such as diarrhoea. VIP increasing strainsalso improve intestinal epithelial barrier integrity. Such strains arein particular useful and more efficacious than existing strains inprevention and/or treatment of IBS and/or constipation and otherdisorders associated with a decreased function of the ENS.

Increasing cholinergic phenotype, in particular the expression of ChATneurons is of therapeutic interest in GI tract pathologies associatedwith inhibition of colonic transit. Using lactic acid bacteria orbifidobacteria selected to have an increasing effect on enhancingcholinergic expression, i.e. ChAT, in neurons are of therapeuticalinterest for constipated patients, and patients suffering from IBS-C.Therefore, one group of lactic acid bacteria or bifidobacteria strainsof the present invention advantageously increases the number of ChATnerves, which is indicative for an improved effect on intestinalmotility, and especially is beneficial for IBS patients, in particularIBS-C patients, and patients suffering from constipation. This group isreferred to as group B). Good motility requires a high number of ChATnerves, which are responsible for contraction and have a prokineticeffect. In some interesting embodiments for this group the TEER levelsare not decreased, since the IEB function and ability to relax themuscles is preferably not impaired. The subgroup according to theseembodiments is referred to as group B3).

Increasing VIP beneficially improves relaxation of the muscles of the GItract and improves IEB, which is beneficial for patients suffering fromIBS or inflammatory bowel disease (IBD) and also for elderly people,infants, and obese people. For such subjects a good IEB function isbetter if not essential. Although patients suffering from IBS or IBDhave an oversecretion of neuropeptides such as VIP, this is supposed tobe an adaptative response of the ENS to control intestinal inflammation,to re-establish intestinal barrier functions and to increaseneuroprotection. A group of lactic acid producing bacteria strains, inparticular bifidobacteria, of the present invention advantageouslyincreases VIP. This group is referred to as group A). In one embodimentthe ChAT level is not increased. The group according to this embodimentis referred to as group A3). Accordingly the ChAT level can remainsubstantially unchanged or can decrease.

All the herein referred bacterial strains have been deposited, accordingto the Budapest Treaty, before CNCM (“Collection Nationale de Culturesde Microorganismes”, 25 rue du Docteur Roux, Paris) as an Internationaldepositary authority.

Strains found with the screening method were belonging to group B) areDN_(—)154_(—)0067 (CNCM I-4320 filed May 19, 2010), DN_(—)116_(—)0047(CNCM I-4317 filed May 19, 2010) and DN_(—)119_(—)0118 (CNCM I-4279filed Feb. 25, 2010). Strains belonging to group A) are DN_(—)173_(—)010(CNCM I-2494 filed Jun. 20, 2000), DN_(—)156_(—)0032 (CNCM I-4321 filedMay 19, 2010), DN_(—)156_(—)007 (CNCM I-2219 filed May 31, 1999), andDN_(—)121_(—)0304 (CNCM I-4318 filed May 19, 2010). StrainDN_(—)173_(—)010 (CNCM I-2494 filed Jun. 20, 2000) has been disclosed inInternational application WO 02/02800 and strain DN_(—)156_(—)007 (CNCMI-2219 filed May 31, 1999) has been disclosed in Internationalapplication WO 01/01785.

Compositions comprising at least one of these selected strains aretherefore part of the invention. A composition comprising a mix of atleast one strain belonging to group B) and at least one strain belongingto group A) is preferred. Such a mix will advantageously have animproved effect on motility by improving both contractions andrelaxations and additionally have an advantageous effect on IEBfunction.

Thus, according to one aspect the invention concerns a compositioncomprising at least one strain of bacteria, preferably selected from thegroup consisting of lactobacilli and bifidobacteria, for use in:

A) increasing vaso-active intestinal peptide (VIP) levels of the entericnervous system, or

B) increasing Choline AcetylTransferase ImmunoReactive neurones (ChAT)levels of the enteric nervous system, or

C) decreasing ChAT levels of the enteric nervous system.

According to one aspect the invention concerns a composition comprisingat least one strain of bacteria selected from the group consisting ofthe following strains:

DN_(—)156_(—)0032 (CNCM I-4321 filed May 19, 2010) [A)-A3)],

DN_(—)156_(—)007 (CNCM I-2219 filed May 31, 1999) [A)-A3)],

DN_(—)121_(—)0304 (CNCM I-4318 filed May 19, 2010) [A)-A3)],

DN_(—)116_(—)0047 (CNCM I-4317 filed May 19, 2010) [B)-B3)],

DN_(—)154_(—)0067 (CNCM I-4320 filed May 19, 2010) [B)-B3)], and

DN_(—)119_(—)0118 (CNCM I-4279 filed Feb. 25, 2010) [B)-B3],

for use in:

-   -   treatment and/or prevention of an intestinal disorder,        preferably treatment and/or prevention of an intestinal motility        disorder, or    -   treatment and/or prevention of a disorder selected form the        group consisting of constipation and IBS-C,    -   treatment and/or prevention of a disorder selected from the        group consisting of diarrhoea, intestinal infection, IBS-D,        IBS-PI, and IBD, or    -   treatment and/or prevention of a disorder selected from the        group consisting of IBS and IBD, or    -   treatment and/or prevention of disorders found in elderly        people, infants, or obese people.

According to one aspect the invention concerns a composition comprisingat least one strain of bacteria selected from the group consisting ofthe following strains:

DN_(—)156_(—)0032 (CNCM I-4321 filed May 19, 2010) [A)-A3)],

DN_(—)156_(—)007 (CNCM I-2219 filed May 31, 1999) [A)-A3)],

DN_(—)121_(—)0304 (CNCM I-4318 filed May 19, 2010) [A)-A3)],

DN_(—)116_(—)0047 (CNCM I-4317 filed May 19, 2010) [B)-B3)],

DN_(—)154_(—)0067 (CNCM I-4320 filed May 19, 2010) [B)-B3)], and

DN_(—)119_(—)0118 (CNCM I-4279 filed Feb. 25, 2010)[B)-B3)],

for use in administration to subjects suffering from a disorder selectedfrom the group consisting of:

-   -   constipation, IBS-C,    -   diarrhoea, intestinal infection, IBS-D, IBS-PI, IBD,    -   IBS, and    -   disorders found in elderly people, infants, or obese people.

According to one aspect the invention concerns a composition asmentioned above for use in improving gastro-intestinal motility,improving intestinal peristalsis and/or decreasing intestinalpermeability.

According to one aspect the invention concerns new strains of bacteriaselected from the group consisting of:

-   -   DN_(—)156_(—)0032 (CNCM I-4321 filed May 19, 2010) [A)-A3)],    -   DN_(—)121_(—)0304 (CNCM I-4318 filed May 19, 2010) [A)-A3)],    -   DN_(—)116_(—)0047 (CNCM I-4317 filed May 19, 2010) [B)-B3)], and    -   DN_(—)154_(—)0067 (CNCM I-4320 filed May 19, 2010) [B)-B3)].

According to one aspect the invention concerns compositions comprisingthe new strains.

According to one aspect the invention concerns a composition comprising:

-   -   at least one strain of bacteria selected from the group        consisting of lactobacilli and bifidobacteria that B) increases        ChAT levels in the enteric nervous system, and    -   at least one strain of bacteria selected from the group        consisting of lactobacilli and bifidobacteria that A) increases        vaso-active intestinal peptide (VIP) levels in the enteric        nervous system.

According to one aspect the invention concerns a method of selectingstrains of bacteria, said method comprising the steps of:

a) Arranging a coculture of intestinal epithelial cells and entericneuronic cells, wherein said intestinal epithelial cell are present as amonolayer and wherein said enteric neuronic cells are present at thebasoluteral side of the monolayer,

b) Adding strains of bacteria to the apical or luminal side of themonolayer of intestinal epithelial cells, preferably in an amount ofabout 4 to 400 bacterial cells per epithelial cell,

c) Incubating the coculture with the strain of lactic acid bacteria,

d) Preferably isolating the neuronic cells,

e) Measuring the amount of VIP, ChAT, substance P, Nitrogen Oxidenerves, ATP and/or pituitary adenylate cyclase activating peptide(PACAP) produced by the neuronic cells and optionally additionally theTransEpithelial Electrical Resistance (TEER) of the intestinalepithelial cells layer.

In this method, the strain of bacteria preferably belongs to the groupconsisting of lactobacilli and bifidobacteria.

DETAILED DESCRIPTION Definitions

In the present application the use of a compound or a composition isintended to cover the use itself, optionally with the connectedintention, but also any communication associated to the compound orcomposition with commercial or legal consequences, for exampleadvertisement, instructions or recommendation on the package of thecompositions, instructions or recommendation on commercial support suchas leaflets, brochures, posters, documentation filed in support toregulatory registrations for safety purpose, efficacy purpose, orconsumer protection, for example at administrations such as EFSA inEurope.

In the present application groups of strains refer to strains thatexhibit a specific property or set of properties. A specific strain canthus pertain to several groups. In the present application the term “or”is not exclusive.

In the present application a property such as VIP and/or ChAT isconsidered as substantially unchanged compared to a control if thevariation does not exceed 10%, preferably 1% compared to the control.

Preferred Embodiments

In preferred embodiments, the composition is for use in:

A3) increasing VIP, provided that ChAT is not increased, or

B3) increasing ChAT, provided that VIP is not increased, ElectricalResistance (TEER) of the intestinal epithelial cells layer being notdecreased or

C3) decreasing ChAT, provided that VIP is not decreased, or

C2) decreasing ChAT and decreasing VIP.

For example the composition of the invention can be used in:

-   -   treatment and/or prevention of an intestinal disorder,        preferably treatment and/or prevention of an intestinal motility        disorder, or    -   treatment and/or prevention of a disorder selected form the        group consisting of constipation and IBS-C, or    -   treatment and/or prevention of a disorder selected from the        group consisting of diarrhoea, intestinal infection, IBS-D,        IBS-PI, and IBD, or    -   treatment and/or prevention of a disorder selected from the        group consisting of IBS and IBD, or    -   treatment and/or prevention of disorders found in elderly        people, infants, and obese people.        In especially preferred embodiments, the compositions can:

A) increase VIP levels of the enteric nervous system, and be used intreatment and/or prevention of an intestinal disorder, preferablytreatment and/or prevention of an intestinal motility disorder, or

B) increase ChAT levels of the enteric nervous system, and be used intreatment and/or prevention of a disorder selected form the groupconsisting of constipation and IBS-C, or

C) decrease ChAT levels of the enteric nervous system, and be used intreatment and/or prevention of a disorder selected from the groupconsisting of diarrhoea, IBS-D, IBS-PI, IBD.

The strain of bacteria can for example be selected from the groupconsisting of the following strains:

-   -   DN_(—)173_(—)010 (CNCM I-2494 filed Jun. 20, 2000) [A)-A3)],    -   DN_(—)156_(—)0032 (CNCM I-4321 filed May 19, 2010) [A)-A3)],    -   DN_(—)156_(—)007 (CNCM I-2219 filed May 31, 1999) [A)-A3)],    -   DN_(—)121_(—)0304 (CNCM I-4318 filed May 19, 2010) [A)-A3)],    -   DN_(—)116_(—)0047 (CNCM I-4317 filed May 19, 2010) [B)-B3)],    -   DN_(—)154_(—)0067 (CNCM I-4320 filed May 19, 2010) [B)-B3)], and    -   DN_(—)119_(—)0118 (CNCM I-4279 filed Feb. 25, 2010) [B)-B3)].        In one embodiment the strain of bacteria is selected from the        group consisting of the following strains:    -   DN_(—)156_(—)0032 (CNCM I-4321 filed May 19, 2010) [A)-A3)],    -   DN_(—)156_(—)007 (CNCM I-2219 filed May 31, 1999) [A)-A3)], and    -   DN_(—)121_(—)0304 (CNCM I-4318 filed May 19, 2010) [A)-A3)],

and the composition is for use in:

-   -   treatment and/or prevention of a disorder selected from the        group consisting of IRS and IBD, or    -   treatment and/or prevention of disorders found in elderly        people, infants, or obese people.        In one embodiment the strain of bacteria is selected from the        group consisting of the following strains:    -   DN_(—)116_(—)0047 (CNCM I-4317 filed May 19, 2010) [B)-B3)],    -   DN_(—)154_(—)0067 (CNCM I-4320 filed May 19, 2010) [B)-B3)], and    -   DN_(—)119_(—)0118 (CNCM I-4279 filed Feb. 25, 2010) [B)-B3)],

and the composition for use in treatment and/or prevention of a disorderselected from the group consisting of constipation and IBS-C.

In one embodiment the strain of bacteria is selected from the groupconsisting of the following strains:

-   -   DN_(—)156_(—)0032 (CNCM I-4321 filed May 19, 2010) [A)-A3)],    -   DN_(—)156_(—)007 (CNCM I-2219 filed May 31, 1999) [A)-A3)], and    -   DN_(—)121_(—)0304 (CNCM I-4318 filed May 19, 2010) [A)-A3)],

and the composition is for use in:

A) increasing vaso-active intestinal peptide (VIP) levels of the entericnervous system, preferably for use in A3) increasing VIP, provided thatChAT is not increased.

In a particular embodiment of this embodiment, the composition is foruse in:

-   -   treatment and/or prevention of a disorder selected from the        group consisting of IBS and IBD, or    -   treatment and/or prevention of disorders found in elderly        people, infants, or obese people.        In one embodiment the strain of bacteria is selected from the        group consisting of the following strains:    -   DN_(—)116_(—)0047 (CNCM I-4317 filed May 19, 2010) [B)-B3)],    -   DN_(—)154_(—)0067 (CNCM I-4320 filed May 19, 2010) [B)-B3)], and    -   DN_(—)119_(—)0118 (CNCM I-4279 filed Feb. 25, 2010) [B)-B3)],

and the composition is for use in:

B) increasing ChAT levels of the enteric nervous system, preferably 13)increasing ChAT, provided that VIP is not increased.

In a particular embodiment of this embodiment, the composition is foruse in treatment and/or prevention of a disorder selected form the groupconsisting of constipation and IBS-C.

Further Details of Strains of Bacteria

As mentioned above the composition comprises at least one or twospecific strains of bacteria, preferably lactic acid bacteria. They arepreferably selected from the group consisting of the genus Lactobacillusand Bifidobacterium, Lactococcus and Streptococcus. The said specificstrain of bacteria were found to be capable to affect VIP levels and/orto affect ChAT nerve levels in a coculture model representing theinteraction between the intestine and the ENS.

A coculture model, described in more detail below, was used to in vitroselect strains of lactic acid bacteria or bifidobacteria with theseproperties. 102 strains belonging to the genera Lactobacillus,Streptococcus or Bifidobacterium were screened.

Group 1) strains with increased effect on ChAT will typically improveintestinal motility. Group B3) strains with increased effect on ChAT,excluding strains wherein VIP is not increased (i.e. VIP issubstantially unchanged or VIP is decreased) represent a specificpreferred embodiment. Strains of these groups are typically beneficialfor treatment and/or prevention of a disorder selected form the groupconsisting of constipation and IBS-C. This might be of furtherparticular interest for treatment and/or prevention of disorders foundin elderly people, which can often suffer from constipation. Concerningincreasing motility, strains increasing ChAT expression would befavoured. This property appears to be very rare. Interestingly, onlythree strains were found to increase statistically ChAT. These arereferred to group B) or group 133) strains. Group B) or group B3)strains comprise strains DN_(—)119_(—)0118 (CNCM I-4279 filed Feb. 25,2010), DN_(—)154_(—)0067 (CNCM I-4320 filed May 19, 2010) andDN_(—)116_(—)0047 (CNCM I-4317 filed May 19, 2010). Such strainsbeneficially improve motility, especially contractions. It is preferredthat Electrical Resistance (TEER) of the intestinal epithelial cellslayer be not decreased. Such a property can be indicative of a suitablebarrier function. Strain DN_(—)119_(—)0118 (CNCM I-4279 filed Feb. 25,2010) significantly decreased VIP levels, whereas the other two strainsdid not have a significant effect on VIP levels. Since a decrease in VIPmay have an adverse effect on IEB it was examined with an in vitro modelwith a monolayer of intestinal epithelial cells whether incubation ofthis strain resulted in a decrease transepithelial electrical resistance(TEER). This turned out not to be the case, indicating that the IEBfunction is not impaired.

Concerning the relaxation of the muscles and reinforcement of IEBfunction, which is beneficial in IBS and IBD patients, strains would befavoured that increase VIP expression (which would have in additionanti-inflammatory effects). Such strains are referred to as group A)strains. In a preferred embodiment group A) strains do not increase ChATexpression (i.e. ChAT is substantially unchanged or ChAT is decreased).Such strains are referred to as group A3) strains. Strains of thesegroups are typically beneficial to treatment and/or prevention of adisorder selected from the group consisting of IBS and IBD, or totreatment and/or prevention of disorders found in elderly people,infants, or obese people. Group A) or group A3) strains comprise strainsDN_(—)173_(—)010 (CNCM I-2494 filed Jun. 20, 2000), DN_(—)156_(—)0032(CNCM I-4321 filed May 19, 2010), DN_(—)156_(—)007 (CNCM I-2219 filedMay 31, 1999), DN_(—)121_(—)0304 (CNCM I-4318 filed May 19, 2010). It isinteresting to note that all the strains having this property arebifidobacteria except one: DN_(—)121_(—)0304 (CNCM I-4318 filed May 19,2010). Furthermore, using another in vitro model with a T84 monolayerand the transepithelial electric resistance (TEER) especially strainsDN_(—)173_(—)010 (CNCM I-2494 filed Jun. 20, 2000), DN_(—)156_(—)007(CNCM I-2219 filed May 31, 1999), DN_(—)121_(—)0304 (CNCM I-4318 filedMay 19, 2010) and DN_(—)156_(—)0032 (CNCM I-4321 filed May 19, 2010) ofgroup A) or A3) were found to have a protective effect on the IEB in thepresence of LPS. Therefore, these particular strains are especiallypreferred.

According to one embodiment the strains allow decreasing ChAT levels ofthe enteric nervous system. The corresponding group of strains isreferred to as group C). In a particular embodiment the strains allowdecreasing ChAT, provided that VIP is not decreased (i.e. VIP issubstantially unchanged or VIP is increased). This group is referred toas group C3). In a particular embodiment the strains allow decreasingChAT with decreasing VIP. These strains are referred to as group C2).Strains of group C3) are typically beneficial to treatment and/orprevention of a disorder selected form the group consisting ofdiarrhoea, IBS-D. This might be of further particular interest fortreatment and/or prevention of disorders found in elderly people, whichcan often suffer from diarrhoea. Strains of group C2) are typicallybeneficial to treatment and/or prevention of a disorder selected fromthe group consisting of diarrhoea, IBS-D, IBS-PI, and IBD. This might beof further particular interest for treatment and/or prevention ofdisorders found in elderly people which can often suffer from suchconditions, especially diarrhoea.

The present invention also encompasses the use of the above mentionedstrains, but also mutant strains or genetically transformed strainsderived from any one of the parent strains still having activity on VIPand effecting ChAT nerves. These mutant or genetically transformedstrains can be strains wherein one or more endogenous gene(s) of theparent strain has (have) been mutated, for instance to modify some ofits metabolic properties (e.g. its ability to ferment sugars, itsresistance to acidity, its survival to transport in the gastrointestinaltract, its post-acidification or its metabolite production). They canalso be strains resulting from the genetic transformation of the parentstrain by one or more gene(s) of interest, for instance in order to giveto said strain additional physiological features, or to allow it toexpress proteins of therapeutic or vaccinal interest that one wishes toadminister through said strains.

Preferably a mix of at least one strain belonging to group B),preferably group B3), and at least one strain belonging to group A),preferably group A3), is used. Such a mix will advantageously have animproved effect on motility as well as on IEB.

Co-Culture Model and Screening Assay

In one embodiment the present invention relates to a method of selectingstrains of lactic acid bacteria, said method comprising the steps of:

a) Using a coculture of intestinal epithelial cells and enteric neuroniccells, wherein the intestinal epithelial cells are present as amonolayer and wherein the enteric neuronic cells are present at thebasolateral side of the monolayer,

b) Adding lactic acid bacteria or the apical or luminal side of themonolayer, preferably in an amount of about 4 to 400 bacterial cells perepithelial cell,

c) Incubating the coculture with the lactic acid bacteria,

d) Preferably isolating the neuronic cells, and

e) Measuring the amount of at least one neurotransmitter selected fromthe group consisting of VIP, ChAT, substance P and Nitrogen Oxide, ATP,PACAP produced by the neuronic cells, and optionally additionally theTransEpithelial Electrical Resistance (TEER) of the intestinalepithelial cells layer.

In agreement with the peristaltic reflex the ENS contains hardwiredcircuits that consist of ascending excitatory motor neurons that releaseacetylcholine and substance P, which contracts smooth muscle throughmuscarinic receptors, and of descending inhibitory neurons that releasea cocktail of transmitters, like NO, ATP, VIP and PACAP, all of whichinhibit the circular muscle.

Cell Culture

A suitable way to set up the coculture with a monolayer of polarizedintestinal epithelial cells is given in example 1 and is also describedin J. Chevalier et al, 2008, J. Physiol. 586 1963-1975.

All intestinal epithelial cell cultures forming monolayers are suitable,such as Caco-2, T84, HT29, and TC7. Preferably T84 cells are used.

As primary enteric nerve system cells, preferably cells are isolatedfrom non-human mammalian foetuses, preferably rodents, more preferablyrats.

Preferably the bacteria strains tested are grown to late exponentialphase in a suitable growth medium and washed. Preferably the bacteriaare added to the apical side of the coculture at an amount of 4 to 400bacteria/epithelial cell, more preferably 10 to 100 bacteria/epithelialcell, even more preferably 30 to 50 bacteria/epithelial cell.Preferably, as a control, no bacteria are added. Preferably, as apositive control 1 mM butyrate or 40 mM KCl is used.

Preferably the incubation step is performed at about 37° C. Preferablythe incubation step takes 1 to 72 h, more preferably 2 to 36 h, evenmore preferably 4 to 12 h.

Preferably after co-incubation, the compartment containing epithelialcells and bacteria is removed and primary neuronal cells are incubatedfor 12 to 48 h, more preferably for 20 to 28 h in a humidified incubatorcontaining 5% CO₂.

Preferably the amount of ChAT nerves versus total nerves is measuredusing immunohistochemical staining, using anti-neurone specific enolase(NSE) to count the total number of neurones and anti-choline acetyltransferase to count the ChAT nerves.

Preferably VIP is determined by ELISA after collecting the neuronalcells and extracting the proteins with the presence of a proteaseinhibitor cocktail.

Further Details about Compositions

The invention encompasses compositions with strains of bacteria whichallow the above referenced uses or properties. The invention alsoencompasses compositions comprising one or more of the following strains(encompassing mutants or genetically transformed strains derivedthereof):

-   -   DN_(—)156_(—)0032 (CNCM I-4321 filed May 19, 2010) [A)-A3)],    -   DN_(—)156_(—)007 (CNCM I-2219 filed May 31, 1999) [A)-A3)],    -   DN_(—)121_(—)0304 (CNCM I-4318 filed May 19, 2010) [A)-A3)],    -   DN_(—)116_(—)0047 (CNCM I-4317 filed May 19, 2010) [B)-B3)],    -   DN_(—)154_(—)0067 (CNCM I-4320 filed May 19, 2010) [B)-B3)], and    -   DN_(—)119_(—)0118 (CNCM I-4279 filed Feb. 25, 2010) [B)-B3)],

for use in:

-   -   treatment and/or prevention of an intestinal disorder,        preferably treatment and/or prevention of an intestinal motility        disorder, or    -   treatment and/or prevention of a disorder selected form the        group consisting of constipation and IBS-C, or    -   treatment and/or prevention of a disorder selected from the        group consisting of diarrhoea, intestinal infection, IBS-D,        IBS-PI, and IBD, or    -   treatment and/or prevention of a disorder selected from the        group consisting of IBS and IBD, or    -   treatment and/or prevention of disorders found in elderly        people, infants, or obese people,

typically when administered in vivo to a subject.

In the compositions of the invention, said strains can be used in theform of whole bacteria which may be living or not. Alternatively, theycan be used in the form of a bacterial lysate or in the form ofbacterial fractions; the bacterial fractions suitable for this use canbe chosen, for example, by testing their properties of alleviating theeffects on VIP levels and levels of ChAT nerves of the coculture modeldescribed in the present invention. Preferably the bacterial cells arepresent as living, viable cells.

The compositions of the invention can be in any form suitable foradministration, in particular oral administration. This includes forinstance solids, semi-solids, liquids, and powders. Liquid compositionsare generally preferred for easier administration, for instance asdrinks.

The composition can for example comprise at least 10⁵, preferably atleast 1×10⁶, cfu per g dry weight, of at least one strain of bacteria,preferably of strains of bacteria as mentioned above. These arepreferably selected from the group consisting of lactobacilli andbifidobacteria.

When the bacteria are in the form of living bacteria, the compositionmay typically comprise 10⁵ to 10¹³ colony forming units (cfu),preferably at least 10⁶ cfu, more preferably at least 10⁷ cfu, stillmore preferably at least 10⁸ cfu, and most preferably at least 10⁹ cfuper g dry weight of the composition. In the case of a liquidcomposition, this corresponds generally to 10⁴ to 10¹² colony formingunits (cfu), preferably at least 10⁵ cfu, more preferably at least 10⁶cfu, still more preferably at least 10⁷ cfu, and most preferably atleast 10⁹ cfu/ml.

Examples of the compositions of the invention are nutritionalcompositions, including food products and in particular dairy products.

The composition can be for example a dairy product, preferably afermented dairy product. The administration in the form of a fermenteddairy product has the additional advantage of low lactose levels, whichis further beneficial for IBS. Optionally, other strains of lactic acidbacteria may be present. The fermented product can be present in theform of a liquid or present in the form of a dry powder obtained bydrying the fermented liquid. Preferably the fermented product is a freshproduct. A fresh product, which has not undergone severe heat treatmentsteps, has the advantage that bacterial strains present are in theliving form. Preferably the fermented product is a dairy product, morepreferably fermented milk and/or fermented whey. Preferably thenutritional composition is yoghurt, or fermented milk in set, stirred ordrinkable form. Preferably the fermented product is a cheese. Preferablythe fermented product is a fermented vegetable, such as fermented soy,cereals and/or fruits in set, stirred or drinkable forms.

Preferably the present nutritional composition is a baby food, an infantmilk formula or an infant follow-on formula. Preferably the presentcomposition is a nutraceutical or a pharmaceutical product, anutritional supplement or medical food.

Nutritional compositions of the invention also include food supplements,and functional food. A “food supplement” designates a product made fromcompounds usually used in foodstuffs, but which is in the form oftablets, powder, capsules, potion or any other form usually notassociated with aliments, and which has beneficial effects for one'shealth. A “functional food” is an aliment which also has beneficialeffects for one's health. In particular, food supplements and functionalfood can have a physiological effect—protective or curative—against adisease, for example against a chronic disease.

A composition comprising a mix of at least one strain of lactic acidbacterium or bifidobacterium increasing ChAT nerves and at least onestrain of lactic acid bacterium or bifidobacterium increasing VIP levelsis preferred. Such a mix will advantageously have an improved effect onmotility as well as on IEB.

A mix of at least one strain belonging to group B), preferably B3), andat least one strain belonging to group A), preferably A3), is preferred.Such a mix will advantageously have an improved effect on motility aswell as on IEB.

Therefore the present invention also relates to compositions comprising:

-   -   at least one strain of bacteria selected from the group        consisting of the following strains:

DN_(—)116_(—)0047 (CNCM I-4317 filed May 19, 2010) [B)-B3)],

DN_(—)154_(—)0067 (CNCM I-4320 filed May 19, 2010) [B)-B3)], and

DN_(—)119_(—)0118 (CNCM I-4279 filed Feb. 25, 2010)[B)-B3)]; and

-   -   at least one strain of bacteria selected from the group        consisting of the following strains:

DN_(—)173_(—)010 (CNCM I-2494 filed Jun. 20, 2000) [A)-A3)],

DN_(—)156_(—)0032 (CNCM I-4321 filed May 19, 2010) [A)-A3)],

DN_(—)156_(—)007 (CNCM I-2219 filed May 31, 1999) [A)-A3)], and

DN_(—)121_(—)0304 (CNCM I-4318 filed May 19, 2010) [A)-A3)].

The compositions of the invention can also comprise one or more otherstrain(s) of lactic acid bacteria, probiotic or not, for instance one ormore bacterial strain(s) selected from the genera Lactobacillus,Lactococcus, Streptococcus, and Bifidobacteria. In particular, this(these) other strain(s) can include one or more strain(s) ofStreptococcus thermophilus, and/or one or more strain(s) ofLactobacillus bulgaricus.

Application

In one embodiment strains of the present invention were found toincrease the number of ChAT nerves. Choline acetyltransfcrase EC 2.3.1.6is an enzyme that is synthesized within the body of a neuron andtransferred to the nerve terminal. The role of choline acetyltransferaseis to join Acetyl-CoA to choline, resulting in the formation of theneurotransmitter acetylcholine. It is used as an immunohistochemicalmarker for motor neurons. The effects on the ChAT nerve result in theimproved contractions resulting in improved peristalsis. Therefore, thestrains and compositions of the present invention able to increase ChATnerves are advantageously administered to improve the ENS, to improve orenhance peristalsis, to improve intestinal motility and/or to decreasethe gastro-intestinal transit time. Increasing cholinergic phenotype isof therapeutic interest in GI pathologies associated with inhibition ofcolonic transit. In particular, various studies have shown that slowtransit could be associated with a reduced expression of ChAT neurons.In particular, (i) severely constipated patients generally have a loweramount of ChAT nerves, (ii) the production of myenteric AChsignificantly decreased both during the course of infection and postinfection (PI), (iii) during aging a reduction of the proportion ofcholinergic neurons has been reported.

In this context, using strains of bacteria, preferably lactic acidbacteria or bifidobacteria to enhance cholinergic expression in neuronscould be of future therapeutically interest for severely constipatedpatient and IBS-C. Therefore, the strains and compositions of thepresent invention are advantageously administered to patients sufferingfrom IBS-C, and/or constipation. Strains that are most useful are thegroup B) or B3) strains mentioned above.

In one particular embodiment the strains and compositions of the presentinvention are used by or for elderly people. Elderly people in thepresent invention are defined as human with an age above 65 years,preferably above 70 years, preferably above 75 years, preferably above80 years, preferably above 85 years. Elderly people typically havedecreased number of ChAT neurons in the enteric nervous system locatedin the colon, most preferably in the transversal colon. Therefore, thestrains and compositions of the present invention are advantageouslyadministered to treat and/or prevent IBS, preferably IBS-C, and/orconstipation for elderly people. Strains able to increase ChAT nervesare group B), such as strain DN_(—)154_(—)0067 (CNCM I-4320 filed May19, 2010), DN_(—)116_(—)0047 (CNCM I-4317 filed May 19, 2010) andDN_(—)119_(—)0118 (CNCM I-4279 filed Feb. 25, 2010). They preferably donot decrease VIP, since VIP is necessary for a good IEB function and forrelaxation of the GI tract, another important part of the GI tractmotility such as peristalsis. The strains meeting this criterion wereDN_(—)154_(—)0067 (CNCM I-4320 filed May 19, 2010), andDN_(—)116_(—)0047 (CNCM I-4317 filed May 19, 2010). However, also strainDN_(—)119_(—)0118 (CNCM I-4279 filed Feb. 25, 2010) did not negativelyaffect IEB function as determined by TEER experiments, as TEER did notdecreased.

In one embodiment the strains of the present invention were found toincrease the levels of VIP. With respect to the digestive system, VIPinduces smooth muscle relaxation (lower oesophageal sphincter, stomach,gallbladder), stimulates secretion of water into pancreatic juice andbile, and causes inhibition of gastric acid secretion and absorptionfrom the intestinal lumen. Its role in the intestine is to greatlystimulate secretion of water and electrolytes, as well as dilatingintestinal smooth muscle, dilating peripheral blood vessels, stimulatingpancreatic bicarbonate secretion, and inhibiting gastrin-stimulatedgastric acid secretion. These effects work together to increasemotility. Therefore this finding is indicative for these strains to havean improved effect on intestinal motility, in particular the relaxationpart of motility. VIP also beneficially increases IEB function.Therefore, the strains and compositions of the present invention areadvantageously administered to improve the ENS, to improve or enhanceperistalsis, to decrease permeability, to improve intestinal motilityand/or to decrease the gastro-intestinal transit time. Therefore, thestrains and compositions of the present invention are advantageouslyadministered for use in or to patients suffering from:

-   -   treatment and/or prevention of an intestinal disorder,        preferably treatment and/or prevention of an intestinal motility        disorder, or    -   treatment and/or prevention of a disorder selected form the        group consisting of constipation and IBS-C, or    -   treatment and/or prevention of a disorder selected from the        group consisting of diarrhoea, intestinal infection, IBS-D,        IBS-PI, and IBD, or    -   treatment and/or prevention of a disorder selected from the        group consisting of IBS and IBD, or    -   treatment and/or prevention of disorders found in elderly        people, infants, and obese people.        Strains that are most useful are the group A) or A3) strains        mentioned above.        Details or advantages of the present invention can be found in        the non limitative examples below.

EXAMPLES Example 1 Screening of Probiotics in a Co-Culture ModelInvolving Epithelial Cells and Enteric Neuronal Cells Cell Culture

Pregnant Sprague-Dawley rats were purchased (CERJ, Le Genest St Isle,France and Janvier-Breeding Center, Belgium) and killed by an overdoseof CO₂ followed by severing the carotid arteries. The embryos (35-45 perisolation from 3 pregnant rats) were removed and killed by decapitation.The small intestines of embryos were removed and finely diced in HBSS(Sigma, France). Tissue fragments were collected in 5 mil of medium(DMEM-F₁₂ 1:1 medium) and digested at 37° C. for 15 min in 0.1% trypsin(Sigma). The trypsin reaction was stopped by adding 10 ml of mediumcontaining 10% foetal calf serum and then treated by DNAse 1 (0.01%,Sigma) for 10 min at 37° C. After triturating with a 10 ml pipette,cells were centrifuged at 750 r.p.m. for 10 min. Cells were counted andthen seeded at a density of 2.4×10⁵ cells/cm² on 24 well platespreviously coated for 6 h with a solution of gelatine (0.5%, Sigma) insterile phosphate buffered saline (PBS). After 24 h, the medium wasreplaced with a serum free medium (DMEM-F12 1:1 containing 1% of N−2supplement (Life technologies, France). Cells were maintained in culturefor 14 days to obtain primary culture of enteric nervous system (ENS).Half of the medium was replaced every other day. At 14 days the primaryneuronal cells were ready for the establishment of the co-culture model.

T84 cell line (EATCC) was cultured in DMEM-F12 (1:1, GIBCO) supplementedwith 10% heat inactivated FBS and 50 IU/ml penicillin and 50 μg/mlstreptomycin. Cells were seeded in 12-well Transwell® filters (Corning,N.Y. USA) at a density of 2×10⁵ cells/insert and cultured to obtainconfluence.

One day after epithelial cells arrived to confluence, Transwell® filterswere transferred in the 12-well plates seeded at the bottom with entericnervous cells. Epithelial and neuronal cells were co-cultured in themedium for epithelial cells.

Growing of Strains of Bacteria

Bacteria were grown for 16 hrs in TGYH for bifidobacteria andlactobacilli, except for strain DN_(—)173_(—)010 (CNCM I-2494 filed Jun.20, 2000) which was grown on MRS+cysteine medium, washed in PBS twiceand adjusted to 4·10⁸ cfu/ml in order to add consistently the samevolume of bacterial suspension to the filter. The strains were added inthe filter compartment at a MOI of 40 bacteria/epithelial cell. As acontrol, no bacteria were added.

After 8 hrs of co-incubation, the filter compartment containingepithelial cells and bacteria was removed and primary neuronal cellswere incubated for 24 h in a humidified incubator containing 5% CO₂. Inthe control wells neuronal cells where stimulated with 1 mM butyrate and40 mM KCl when ChAT and VIP measurements were performed, respectively.

Immunohistochemical Staining. Measuring ChAT Nerves

After the incubation, immunohistochemistry was performed to detectneuronal cell populations. After cells fixation (in 0.1 M PBS containing4% paraformaldehyde for 1 h at room temperature), cells were washed 3times in PBS, then permeabilized for 30 min in PBS/NaN₃ containing 0.5%Triton X-100 and 4% horse serum. Primary antibody: rabbit anti-neuronespecific enolase (NSE) (1:2000; Biovalley, France) and rabbitanti-choline acetyl transferase was diluted in PBS/NaN₃, 0.5% TritonX-100 and 4% horse serum and incubated overnight at room temperature.After incubation with primary antiserum, cells were washed 3 times withPBS and incubated for 3 h with donkey anti-rabbit IgG conjugated tofluoresceine isothiocyanate (FITC) (1:200 Immunotech, France) and7-amino-4-methyl-coumarin-3-acetate respectively. Specimens were viewedunder an Olympus IXSO fluorescence microscope fitted with white videocamera (Mod. 4910, Cohu Inc, Germany) connected to macintosh computerthrough a frame grabber card (Scion Image, SL Microtest).

VIP Measurements:

For VIP determination, neuronal cells were collected from the 12-wellplates, the proteins were extracted using RIPA lysis buffer (Millipore,France) containing protease inhibitor cocktail (Roche Diagnostics,France) and VIP levels were measured by ELISA (Bachem, Germany).

Results

Differential response of primary enteric neurones on VIP and ChATmarkers following interaction of some of 102 probiotic strains includinglactic acid bacteria and Bifidobacteria are shown in Table 1. Only somestrains belonging to group A3) or B3) or C3) are shown. Additionally itis mentioned that 26 strains were shown to have no significant effect onVIP and ChAT (including strains Bifidobacterium longum NCC 2705 (CNCMI-2618), Lactobacillus rhamnosus GG (ATCC 53103) and Lactobacillus caseiShirota), 11 strains belonging to group C2) were shown to decrease bothVIP and ChAT (including strain Bifidobacterium longum W11 of Alfa-Wass(LMG P-21586)), 10 strains decreased VIP and had no effect on ChAT(including bench mark strains Bifidobacterium infantis UCC 3564,Bifidobacterium longum Bb536, Bifidobacterium animalis spp lactis Bb12(DSM 15954), and Bifidobacterium animalis spp lactis Bi-07 (ATCC SD5220)and 41 strains belonging to group C3) decreased ChAT and had no effecton VIP including strains Lactobacillus johnsonii La1 (CNCM I-1225),Lactobacillus plantarum 299v (DSM 9843) Lactobacillus reuteri SD 2122(ATCC 55730)).

TABLE 1 Effect of incubation with lactic acid bacteria andbifidobacteria on VIP and ChAT levels in a coculture model withepithelial cell monolayer and primary ENS cells. VIP ChAT EstimatedEstimated DN Number difference* p Empiric difference p Empiric Groupspecies (CNCM number) vs control value mean vs control value Mean 1DN_154_0067 (CNCM I- −0.0097 0.9 0.0790 0.2709 0.09 0.1796 4320 filedMay 19 2010) Bifidobacterium bifidum 1 DN_116_0047 (CNCM I- −0.0389 0.70.0397 0.3151 0.10 0.2535 4317 filed May 19 2010) Lactobacillusrhamnosus 1 DN_119_118 (CNCM I- −0.1329 0.09 −0.2221 0.2847 0.02 0.27964279 filed Feb. 25 2010) Lactobacillus acidophillus 2 DN_173_010 (CNCMI- 0.2345 0.01 0.2001 −0.2615 0.05 −0.0825 2494 filed Jun. 20 2000)Bifidobacterium lactis 2 DN_156_0032 (CNCM I- 0.2248 0.01 0.2020 −0.54500.00 −0.5723 4321 filed May 19 2010) Bifidobacterium breve 2 DN_156_007(CNCM I- 0.2715 0.02 0.3552 −0.3632 0.01 −0.1281 2219 filed May 31 1999)Bifidobacterium breve 2 DN_121_0304 (CNCM I- 0.5976 0.00 0.6813 −0.62690.00 −0.3918 4318 filed May 19 2010) Lactobacillus plantarum *Values aregiven as a difference compared to the control, where no bacterialstrains were added.

Although strain DN_(—)119_(—)0118 (CNCM I-4279 filed Feb. 25, 2010)decreases VIP levels, it turned out with a TEER model (Hirotani et al,2008, Yakugaku Zasshi September; 128(9):1363-8) that incubation with thestrain for 4 or 6 h did not significantly reduce TEER values, even inpresence of damage vs. control. In short, bacteria were cultured inTGYH. The culture suspensions were washed with PBS. Subsequently, thebacteria (100 cfu/cell) were added to the apical side of the T84 cellmonolayers. After 2 h incubation, LPS (L4516,—EPEC—0127: B8) was addedon the apical side at 40 ng/ml or not added. Then, after 2 h and 4 hincubation, the TEER value was measured to assess epithelial barrierfunction. All experiments were performed three times independently andin triplicate in presence and in absence of LPS. The value of the T84 att=0 was set at 100%. In the absence of LPS TEER at T4 was 98.7% and atT6 100.2% with strain DN_(—)119_(—)0118 (CNCM I-4279 filed Feb. 25,2010); for T84 alone this was still 100%. In the presence of LPS thecontrol T84 at T4 was 56.2% compared to t=0 and with strainDN_(—)119_(—)0118 (CNCM I-4279 filed Feb. 25, 2010) 47.9%; At T6 the T84control was 46.7% and with strain DN_(—)119_(—)0118 (CNCM I-4279 filedFeb. 25, 2010) 52.2%.

Using this same TEER model especially strain DN_(—)173_(—)010 (CNCMI-2494 filed Jun. 20, 2000), DN_(—)156_(—)007 (CNCM I-2219 filed May 31,1999), DN_(—)121_(—)0304 (CNCM I-4318 filed May 19, 2010) andDN_(—)156_(—)0032 (CNCM I-4321 filed May 19, 2010), all belonging togroup A3), showed good results on the intestinal barrier function asassessed by TEER in presence of LPS. See Table 2.

TABLE 2 TEER results in presence of LPS of selected bacteria showing thebest results TEER T4/ TEER T6/ TEER T0 (%) TEER T0 (%) Signif- EmpiricSignif- Empiric Strain icance mean icance mean T84 control 56.20 46.76DN_173_010 B. lactis *** 71.27 *** 51.03 (CNCM I-2494 filed Jun. 202000) DN_156_007 B. breve *** 70.70 *** 55.87 (CNCM I-2219 filed May 311999) DN_121_0304 L. plantarum *** 65.84 *** 64.67 (CNCM I-4318 filedMay 19 2010) DN_156_0032 B. breve *** 84.44 *** 80.38 (CNCM I-432 filledMay 19 2010) ***p value < 0.05

1. A method of: A. increasing vaso-active intestinal peptide (VIP) levels of the enteric nervous system, or B. increasing Choline AcetylTransferase ImmunoReactive neurones (ChAT) levels of the enteric nervous system, or C. decreasing ChAT levels of the enteric nervous system, comprising administering to a subject a composition comprising at least one strain of bacteria selected from the group consisting of lactobacilli and bifidobacteria, wherein the strain of bacteria is selected from the group consisting of the following strains: DN_(—)173_(—)010 deposited under Accession No. 1-2494 with Collection Nationale De Cultures De Micro-Organismes (CNCM) on Jun. 20, 2000, DN_(—)156_(—)007 deposited under Accession No. 1-2219 with CNCM on May 31, 1999, and DN_(—)119_(—)0118 deposited under Accession No. 1-4279 with CNCM on Feb. 25,
 2010. 2. The method according to claim 1, wherein said composition: increases VIP, provided that ChAT is not increased, or increases ChAT, provided that VIP is not increased, decreases ChAT, provided that VIP is not decreased, or decreases ChAT and decreases VIP.
 3. The method according to claim 1, wherein said composition is administered to the subject for: treatment and/or prevention of an intestinal disorder, or treatment and/or prevention of a disorder selected from the group consisting of constipation and irritable bowel syndrome C (IBS-C), or treatment and/or prevention of a disorder selected from the group consisting of diarrhoea, intestinal infection, IBS-D, IBS-PI, and IBD, or treatment and/or prevention of a disorder selected from the group consisting of IBS and inflammatory bowel Disease (IBD), or treatment and/or prevention of disorders found in elderly people, infants, and obese people.
 4. The method according to claim 1, wherein said composition: A. increases VIP levels of the enteric nervous system, and is used in treatment and/or prevention of an intestinal disorder, or B. increases ChAT levels of the enteric nervous system, and is used in treatment and/or prevention of a disorder selected form the group consisting of constipation and IBS-C, or C. decreases ChAT levels of the enteric nervous system, and is used in treatment and/or prevention of a disorder selected from the group consisting of diarrhoea, IBS-D, IBS-PI, IBD.
 5. The method according to claim 1, wherein said composition: A3 increases VIP provided that ChAT is not increased, and is administered to a subject for: treatment and/or prevention of a disorder selected from the group consisting of IBS and IBD, or treatment and/or prevention of disorders found in elderly people, infants, or obese people.
 6. The method according to claim 1, wherein said composition: B3. increases ChAT, provided that VIP is not increased, and is administered to a subject for treatment and/or prevention of a disorder selected form the group consisting of constipation and IBS-C.
 7. The method according to claim 1, wherein said composition: C3. decreases ChAT, provided that VIP is not decreased, and is administered to a subject for treatment and/or prevention of a disorder selected form the group consisting of constipation and IBS-C.
 8. The method according to claim 1, wherein said composition: C2. decreases ChAT and decreases VIP, and is administered to a subject for treatment and/or prevention of a disorder selected from the group consisting of diarrhoea, intestinal infections, IBS-D, IBS-PI, and IBD. 9-17. (canceled)
 18. The method according to claim 3, wherein said composition is administered to the subject for improving gastro-intestinal motility, improving intestinal peristalsis and/or decreasing intestinal permeability. 19-26. (canceled)
 27. The method according to claim 1, wherein the composition is a fermented milk product.
 28. The method according to claim 27, wherein the fermented milk product is a yogurt. 